Our long term goal is to establish simple biochemical assays that will allow us i) to assess the mechanical effects of a broad range of physiological and disease-related effectors of muscle force, and ii) to rapidly screen small molecule libraries for new therapeutic modulators of muscle force. Our general hypothesis is that most changes in isometric muscle force, F, have a well-defined biochemical basis. Specifically isometric muscle force, F =Funi.N, is influenced by the number, N, of myosin molecules strongly bound to actin and the average force, Funi, per actin bound myosin, where recent studies indicate that Funi varies proportionally with the actin- myosin binding energy, G. The effects of mutations, accessory proteins, and small molecules on the biochemical determinants of isometric muscle force, deltaG and N, can be measured directly in a test tube, suggesting that high throughput screens (HTS) for effectors of muscle force are possible. Using biophysical and biochemical techniques, we have developed several assays that demonstrate the feasibility of a HTS for effectors of muscle force. In this proposal we will (Aim 1) establish a HTS for modulators of muscle force, and we will (Aim 2) verify that these screens are accurate predictors of changes in F, using both in vitro force assays and muscle mechanics experiments. This proposal relies on our laboratory's combined expertise in muscle physiology/biophysics, state-of-the-art biophysical techniques, and theoretical models of muscle mechanochemistry. The HTS for effectors of muscle force developed in this proposal will dramatically accelerate characterization of a wide range of known and suspected modulators of muscle force and will provide a powerful tool for discovering small molecular inhibitors and activators of muscle force. PUBLIC HEALTH RELEVANCE. Fine control of muscle force is central to the normal function, adaptation, and development of most musculoskeletal and organ systems, and disrupting this mechanical balance can lead to a variety of diseases, such as hypertension resulting in cardiac failure and airway hyper-responsiveness associated with asthma. Developing approaches for rapidly assessing the effects of new compounds on muscle mechanics will allow us to rapidly discover new therapies through which we can help muscles in disease states regain their normal mechanical function.